Tsr and chemotaxis mediate efficient pathogen invasion of colonic tissue.

A-B. Overview of the role of Tsr in chemotactic responses and premise of this study. C. Experimental design of colonic explant infections. See Materials & Methods for experiment details such as tissue dimensions. D. Serine (presumed to be nearly 100% LSer, see Materials & Methods) and indole content of liquid human fecal treatments, as measured by mass spectrometry. E-I. Competitive indices (CI) of colony-forming units (CFUs) recovered from co-infected swine explant tissue, either from the total homogenate (open box and whiskers plots), or from tissue washed with gentamicin to kill extracellular and attached cells, which we refer to as the “invaded” intracellular population (checkered box and whisker plots), as indicated. Each data point represents a single experiment of a section of tissue infected with bacteria, and the CI of CFUs recovered from that tissue (n=5). Boxes show median values (line) and upper and lower quartiles, and whiskers show max and min values. Effect size (Cohen’s d) and statistical significance are noted for each experiment in relation to competitive advantage, i.e. deviation from a CI of 1 (not significant, ns; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). See also Figure S1 for disaggregated CFU enumerations for each experimental group prior to CI calculation. Data S1 contains all numerical CFU measurements.

Bacterial Strains

Salmonella Typhimurium exhibits attraction toward liquid human fecal material.

A. Diffusion modeling showing calculated local concentrations in CIRA experiments with liquid human fecal material based on distance from the central injection source. B. Max projections of representative S. Typhimurium IR715 responses to a central source of injected liquid human fecal material. C-E. Bacterial population density over time in response to fecal treatment. The initial uniform population density in these plots is indicated with the blue line (time 0), and the final mean distributions with the red line (time 280 s), with the mean distributions between these displayed as a blue-tored spectrum at 10 s intervals. F-G. Temporal analyses of area under the curve (AUC) or relative number of bacteria within 150 μm of the source. Effect size (Cohen’s d) comparing responses of WT and tsr attraction at 120 s posttreatment is indicated. Data are means and error bars are standard error of the mean (SEM, n=3-5). See also Movie 1, Table S1, and Figure S2.

Fecal indole is insufficient for chemorepulsion but indole in isolation is a strong chemorepellent.

A-E. Dualchannel imaging of chemotactic responses to solubilized human feces by WT S. Typhimurium IR715 (pink) and isogenic mutants or clinical isolate strains (green), as indicated. Shown are max projections at time 295-300 s posttreatment. Data are means and error bars are standard error of the mean (SEM, n=3-5). See also Movies 2-3. F. Representative max projections of responses at 295-300 s of indole treatment. G-H. Quantification of chemorepulsion as a function of indole concentration (n=3-5). I-K. Comparison of WT and tsr mutant responses to L-Ser or indole. See also Fig. S2. L-M. Isothermal titration calorimetry (ITC) experiments with 50 μM S. Typhimurium Tsr ligand-binding domain (LBD) and indole, or with L-Ser in the presence of 500 μM indole. Data are means and error bars are standard error of the mean (SEM, n=3-5). AUC indicates area under the curve. Scale bars are 100 μm. See also Movies 2-3.

S. Typhimurium mediates distinct chemotactic responses based on the ratio of L-Ser to indole.

A-D. Representative max projections of responses to treatments of L-Ser and indole at 295-300 s, as indicated. Scale bars are 100 μm. E. Relative bacterial distribution in response to treatments of 500 μM L-Ser and varying amounts of indole, from panels A-D, with the mean value normalized to 100%. Data are means and error bars are standard error of the mean (SEM, n=3-5). F. Diffusion modeling of local effector concentrations based on sources of 5 mM indole (dark brown), 500 μM L-Ser (blue), 500 μM indole (light brown), and 50 μM indole (yellow) are shown as dashed lines. The approximate local concentration of indole that elicits a transition in chemotactic behavior is highlighted in light blue. G-H. Bacterial growth as a function of L-Ser or indole, at the time point where the untreated culture reaches A600 of 0.5. I-J. Bacterial growth +/-pretreatment with 500 μM indole or L-Ser, and increasing concentrations of indole or L-Ser, as indicated at the time point where the untreated culture reaches A600 of 0.5. Data are means and error bars are standard error of the mean (SEM, n=8-24).

A. Working model for chemotactic compromise orchestrated by Tsr during enteric invasion.

In the intestinal lumen, fecal material contains the nutrient L-Ser, derived from digested food, and bacteriostatic indole, secreted by the gut microbiota. Tsr detects these metabolites as chemoattractant (L-Ser, dark blue arrows) and chemorepellent (indole, brown arrows) signals, respectively. While L-Ser is directly sensed by Tsr via the ligand-binding domain (LBD, yellow), indole is detected through a different mechanism. Tsr integrates these conflicting signals to bias colonization through a behavior termed chemohalation, which promotes taxis toward regions with higher L-Ser-to-indole ratios (large blue and brown arrows, width of arrows denotes the degree of colonization bias). Increased indole levels result in a larger central zone of avoidance and greater bias against colonization. B. During early infection, the attractant-to-repellent ratio in the intestinal lumen is high. S. Typhimurium (gray cells) uses chemotaxis to seek L-Ser (dark blue circles) and other nutrients (light blue circles), promoting proliferation. In the presence of abundant nutrients, the bacteria tolerate exposure to indole (brown circles). C. As infection progresses, nutrient consumption by the proliferating bacteria reduces the attractant-to-repellent ratio in the lumen. D. Once nutrients are sufficiently depleted, the bacteria can no longer tolerate indole exposure, and chemorepulsion from indole increases. This repulsion drives bacteria out of the lumen and into contact with the intestinal mucosa, promoting colonic invasion. In the artificial scenario where only L-Ser is present, chemotaxis offers little advantage for invasion; the bacteria proliferate, but there is no incentive to leave the lumen. Conversely, with only indole present, chemotaxis similarly provides no advantage; although indole repels the bacteria from the lumen, it inhibits bacterial proliferation. In the presence of L-Ser, and likely other attractants, indole repulsion is nullified.

Summary of prior studies related to indole chemotaxis, related to Figure 1.

a Source concentration, or ranges, used in experiment.

b The motility or chemotactic response as reported by the study authors; note that some methods employed may not be able to distinguish between responses as a consequence bacterial growth versus chemotaxis.

c The assay may have limitations in its ability to report on either rapid temporal responses, or localization to or from an effector source.

d In this work, we refer to behaviors of this type as “chemohalation” to be similar to the widely-used terms chemoattraction and chemorepulsion.

Colony-forming units (CFU) recovered from swine colonic explant infections, related to Figure 1.

Shown are disaggregated CFU enumerations for each co-infection experiment with S. Typhimurium IR715 WT and mutant strains following tissue treatments, as indicated. CFUs extracted either using the entire tissue homogenate (total) or extracted from tissue treated with a gentamicin wash to kill exterior non-invaded cells (invaded) are noted separately (see Materials & Methods). Each data point represents CFUs recovered from a single explant experiment (n=5). Box and whisker plots represent the sample median (line), the edges are the upper and lower quartiles, and whiskers extend to the max and min values. The limit of detection was 1 x 105 CFUs. Samples with no CFUs detected are noted on the plot at this limit.

Chemotactic responses to L-Ser or indole, related to Figure 2.

A. CIRA experimental design. B. CIRA microgradient diffusion model, simulated with a source of 1.13 mM A488 dye after 30 s of injection. Image rendered on a pink (100% source concentration)-blue-black (0%) color intensity scale, based on previous work 1. C. CIRA quantification overview, with blue lines corresponding to chemoattraction, null chemotactic response corresponding to gray dashed lines, and chemorepulsion corresponding to brown lines. D-G. Microgradient models of the local effector concentration for treatments with 5 mM L-Ser or indole, respectively, at Δ300 s. Color gradient corresponds to gradually decreasing effector concentrations from yellow to blue. H-I. Representative 10 s max projections of S. Typhimurium IR715 response to sources of L-Ser (strong chemoattraction) or indole (strong chemorepulsion) using CIRA. The glass microcapillary that injects the treatment solution is centered within the field of view. Note that cells contain a plasmid expressing mPlum (see Materials & Methods), and fluorescence data are collected in the far-red channel, so the glass microcapillary is poorly visible.